Project Summary To divide, move, and communicate, cells rely on a dynamic actin cytoskeleton that can rapidly assemble and change. This is achieved by the polymerization of actin monomers into filaments, the construction of filament networks, and the disassembly of these networks back into monomers. Cells maintain a large monomer reserve to meet the demands of actin network assembly. Because of its size, the monomer pool was traditionally considered to be homogeneous. However, recent work by us and others has revealed distinct subgroups of monomers that can drive and modify actin dynamics in ways that profoundly influence cell behavior. These discoveries have shown us that the rules of monomer polymerization are much more complex than previously thought. Addressing this severe knowledge gap in one of the most fundamental aspects of cytoskeletal biology is paramount to understanding how actin functions in cells. It may also finally reveal why defects in monomer regulation are associated with neurodegeneration, inflammatory disorders, cardiac disease and cancer. Most of our current knowledge about actin monomer behavior comes from solution biochemistry. However, these types of experiments cannot reproduce the complex monomer-driven actin dynamics seen in cells. Therefore, we are devising strategies that merge biochemical principles with cellular imaging to reveal how monomeric actin regulates the cytoskeleton in living cells. This includes controlling protein levels with micromolar precision, using quantitative image analysis to extract rate constants and concentrations, and employing computational models to reveal how cellular geometry influences actin dynamics. Additionally, we are developing new tools to describe and quantify actin ultrastructure from super-resolution images. We will use these innovative approaches to address the following fundamental knowledge gaps about monomer-driven actin dynamics: 1) How monomers control the dynamics of complex actin networks, 2) How cell geometry contributes to monomer-regulated actin dynamics, and 3) Novel roles for monomers in the regulation of cellular processes.